The sinoatrial node (SAN), located in the right atrium, serves as the natural cardiac pacemaker and is responsible for initiating each normal heartbeat. SAN dysfunction, characterized by slow and/or irregular pacemaker activity, is common in cardiovascular disease and in the aging population and is a major indicator for cardiac pacemaker implantation. While defects in activity of multiple ion channels have been linked to abnormal SAN cell membrane excitability and SAN dysfunction, little is known about molecular pathways responsible for ion channel membrane targeting. Recent studies from our group have identified a novel role for the actin-associated polypeptide BetaIV spectrin in organizing local signaling domains for membrane ion channels in ventricular myocytes. However, the pathways responsible for organization of analogous domains in SAN cells remain largely unstudied. There is a need to elucidate the mechanisms by which ion channel membrane domains are coordinated to regulate cardiac pacemaking. The objective of this proposal is to define the role of the actin-associated polypeptide Beta-IV spectrin in ion channel targeting and regulation of membrane excitability in SAN cells, and to develop quantitative tools for analysis of SAN cell behavior and susceptibility to dysfunction. My central hypothesis is that Beta-IV spectrin determines normal membrane localization of the mechanosensitive background K+ channel TREK-1 to control SAN cell membrane excitability. Furthermore, I hypothesize that disruption of the Beta-IV spectrin/TREK-1 complex at the sinoatrial node membrane promotes abnormal cell membrane excitability and sinus node dysfunction. My hypotheses will be tested using an innovative combination of experimental and mathematical modeling techniques. I believe that the findings from these studies will identify a pathway for targeting TREK-1 to the sinoatrial node membrane, will develop new analytical/mathematical tools to understand SAN function, and will generate new directions for future research of cell excitability and cardiac pacemaking in normal and diseased hearts.